Oligonucleotides are widely utilized in molecular biological manipulations including DNA sequencing, cycle sequencing, polymerase chain reactions, in vitro mutagenesis, cloning methodologies involving polylinkers and adapters, synthesis of genes by hybridization and ligation of multiple oligonucleotides, and the like methods. Traditionally, oligonucleotides are prepared by chemical synthesis methods de novo each time they are required. Chemical synthesis of oligonucleotides is time consuming and costly.
One approach to DNA sequencing is called "primer walking" which utilizes known sequence information of a target nucleic acid to be sequenced to design a distal primer which is then used to obtain additional, downstream sequence information. Although primer walking is conceptually appealing, because of its simplicity and the ordered nature of the sequence information obtained, this method can be is expensive and time-consuming because after each sequence is determined, a new, customized primer must be chemically synthesized. Because a single oligonucleotide synthesis requires the preparation of more oligonucleotide than is required for the single sequencing step to be performed, material is wasted resulting in excess cost, and synthesis time slows the sequential sequencing steps.
Recently, Studier proposed a strategy to simplify the preparation of unique oligonucleotides in the form of a library of pre-synthesized oligonucleotides representing every possible nucleotide sequence in the size range of oligonucleotides from 8 to 10 nucleotides in length. Studier, Proc.Natl.Acad.Sci., 86:6917-6921 (1989). However, the library poses technical difficulties insofar as the library must contain from 4.sup.8 (65,536) to 4.sup.10 (1,048,576) members, respectively, which is generally considered to be so large as to be unmanageable. In addition, oligonucleotides of 8mer to 10mer length are less preferred sequencing primers than longer oligonucleotides of 12mer to 18mer length.
Szybalski proposed the use of a library of hexameric oligonucleotides comprising every possible combination of nucleotide bases, representing a library having 46 (4,096) members, as a means to reduce the size of the library. Szybalski, Gene, 90:177-178 (1990). Theoretically, pairs of hexamers from the library were proposed to be capable of being individually ligated while hybridized to a template to form 12 nucleotide (nt), 18-nt, or 24-nt oligonucleotides in length, thereby forming every possible nucleotide sequence from a library having 4,096 members. This same approach has been described in U.S. Pat. No. 5,114,639 to Blocker. This approach requires ligation of the hexamer pairs in the presence of template DNA (i.e., DNA molecule to be sequenced).
Accordingly, there continues to exist a need for preparing oligonucleotides suitable for priming PCR, cycle-sequencing and the like reactions without de novo oligonucleotide synthesis or the above-described problems. The present invention meets that need.